Tetrahydrobiopterin (BH4) serves as an essential cofactor for the enzymes that produce nitric oxide, serotonin, dopamine, epinephrine, and norepinephrine. The activities of these enzymes are regulated by the availability of BH4 in the cells. Our long-range objectives of this research are to understand the mechanism that regulates GTP cyclohydrolase I, the first and rate-limiting enzyme in the biosynthetic pathway for BH4. The activity of GTP cyclohydrolase I is controlled by a regulator protein. This key protein is called GTP cyclohydrolase I feedback regulator protein (GFRP), which inhibits the enzyme activity in the presence of BH4 and stimulates it in the presence of phenylalanine through ligand-induced complex formation. During the past funded period we have demonstrated that in both the inhibitory and stimulatory complexes two molecules of GFRP, a pentamer, are physically associated with one molecule of GTP cyclohydrolase I, a decamer. Approximately one molecule of each ligand was found to bind to the respective protein complexes per subunit of the two proteins. Our current working hypothesis is that the catalytic function of GTP cyclohydrolase I is determined by two different conformational states of the protein complexes that are formed by the binding of respective effectors. To test this hypothesis and obtain further information on the regulatory mechanisms of this system, we now propose the following three aims. AIM 1: Define the relationship among enzyme activity, ligand binding, and GFRP binding. AIM II: Determine the three- dimensional structures of GTP cyclohydrolase I, GFRP, the inhibitory complex, and the stimulatory complex, and characterize the conformational transition between these structures. AIM III: Define the functional role of phosphorylation in the GTP cyclohydrolase I-GFRP system and determine the phosphorylation sites of GTP cyclohydrolase I and GFRP. Detailed understanding of the regulatory mechanisms of the GTP cyclohydrolase I/GFRP system will greatly enhance knowledge of metabolism of catecholamines, serotonin, and nitric oxide as well as BH4. The knowledge will provide insight into the altered metabolism of BH4 reported in phenylketonuria, Parkinson's disease, familial dystonia, and several vascular diseases.